CN110699447A - miRNA molecular marker and application thereof in prejudgment of microscopic semen collection operation - Google Patents
miRNA molecular marker and application thereof in prejudgment of microscopic semen collection operation Download PDFInfo
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Abstract
The invention discloses a group of miRNA molecular markers and application thereof in the prejudgment of microscopic semen collection surgery. The miRNA molecular markers comprise miR-34b-3p, miR-34c-3p, miR-3065-3p and miR-4446-3 p. The kit consisting of the marker and the related primer obtained by screening can realize the prejudgment of the microscopic semen-taking operation result, avoid unnecessary traumatic detection and operation on the semen-free patient, save resources, improve efficiency, provide important preoperative reference for an operating doctor, improve the success rate of the operation, have no wound at all, and have simple and convenient operation.
Description
Technical Field
The invention belongs to the technical field of medical examination, and particularly relates to a group of miRNA molecular markers and application thereof in the prejudgment of microscopic semen collection operation.
Background
Infertility accounts for about 15% of the population of the child-bearing age, with male factors accounting for 50% and azoospermia accounting for 10%. At present, the only means for solving the fertility problem of patients without spermatorrhea is microscopic spermatorrhea operation, but the success rate of spermatorrhea operation is closely related to whether spermatogenesis exists in testis, and the only means for judging the state of spermatogenesis in testis is testicular aspiration biopsy, however, the method is invasive test and has certain harm to human body, and the part obtained by aspiration biopsy cannot represent the whole testis due to the characteristic of focal spermatogenesis in testis.
In order to improve the success rate of the microscopic semen collection operation and reduce unnecessary operations and puncture, a method of non-invasive molecular inspection needs to be established, and the problem that the final outcome of the microscopic semen collection operation cannot be predicted clinically at present is solved.
Disclosure of Invention
The invention provides a group of miRNA molecular markers for solving the technical problem that the outcome of microscopic semen collection operation can not be pre-judged clinically at present, and the probability of pre-judging the success rate of the microscopic semen collection operation can be obtained by using the group of miRNA molecular markers for designing a kit and establishing a model equation, so that the success rate of the microscopic semen collection operation is improved, and unnecessary operations and puncture wounds are reduced.
A group of miRNA molecular markers for judging the success rate of microscopical semen collection surgery comprises miR-34b-3p, miR-34c-3p, miR-3065-3p and miR-4446-3 p.
The miRNA molecular marker is applied to preparation of a kit for judging the success rate of microscopic semen collection surgery.
The primer pair for detecting the miRNA molecular marker specifically comprises the following steps:
for miR-34b-3p, the sequence of an upstream primer is shown as SEQ ID NO.9, the sequence of a downstream primer is shown as SEQ ID NO. 13;
for miR-34c-3p, the sequence of an upstream primer is shown as SEQ ID NO.10, the sequence of a downstream primer is shown as SEQ ID NO. 13;
for miR-3065-3p, the sequence of an upstream primer is shown as SEQ ID NO.11, the sequence of a downstream primer is shown as SEQ ID NO. 13;
for miR-4446-3p, the sequence of an upstream primer is shown as SEQ ID NO.12, the sequence of a downstream primer is shown as SEQ ID NO.13, and the sequence is shown as a universal primer.
The primer pair is applied to preparation of a kit for judging the success rate of microscopic semen collection operation.
A kit for judging the success rate of microscopical semen collection operation comprises the primer pair.
Further, reagents commonly used in PCR technology and reagents commonly used in immunohistochemical technology are also included.
Has the advantages that: the kit consisting of the marker and the related primer obtained by screening can realize the prejudgment of the microscopic semen-taking operation result, avoid unnecessary traumatic detection and operation on the semen-free patient, save resources, improve efficiency, provide important preoperative reference for an operating doctor, improve the success rate of the operation, have no wound at all, and have simple and convenient operation.
Drawings
FIG. 1 shows the qPCR results for 40 samples in example 2.
Figure 2 is the correlation results of the four mirnas in example 2.
FIG. 3 is a ROC curve analysis of the accuracy of the expression levels of the four miRNAs in example 2 in predicting spermatogenic function.
Detailed Description
The technical scheme of the invention is realized through a series of experiments and clinical verification. Specifically, the method comprises the following steps: firstly, finding out molecular markers capable of distinguishing the existence/nonexistence of spermatogenesis in testis by gene sequencing of seminal plasma micro RNA (miRNA), wherein four miRNAs can be used as stable and accurate molecular markers, namely mir-34b, mir-34c, mir-3065 and mir-4446; secondly, collecting a large number of patient samples in clinic, carrying out qPCR verification experiments on the four miRNA molecular markers, carrying out machine learning, and finally establishing an equation model, namely miRNA panel; and finally, performing double-blind test by using the four miRNA molecular markers and the designed primers, and verifying the prejudgment probability of the success rate of the microscopic semen collection operation. The specific experimental procedure of the double-blind test is as follows: extracting patient seminal plasma miRNA, carrying out reverse transcription by using a specific stem-loop primer in the kit, then carrying out a pre-amplification experiment (pre-PCR) by using a pre-amplification primer in the kit, then carrying out multiplex digital PCR (multiplex ddPCR) by using a specific probe, obtaining a corresponding fluorescence value and substituting the fluorescence value into a model equation to obtain a probability value, wherein the probability value is the probability for prejudging the success rate of the microscopic semen collection operation.
The invention will be further illustrated with reference to the following specific examples. The experimental procedures used in the following examples are all conventional procedures unless otherwise specified. The materials, reagents and the like used in the following examples are commercially available unless otherwise specified, and techniques not described in detail are performed according to standard methods well known to those skilled in the art. The reagents and the like referred to in this application are commercially available or otherwise publicly available, and are intended to be exemplary only and not exclusive to the present invention. Other suitable tools or biological materials may be substituted, respectively. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention.
Example 1
And (4) screening miRNA molecular markers. Finding out a molecular marker capable of distinguishing the existence of spermatogenesis in testis by seminal plasma micro RNA (miRNA) gene sequencing, and the specific process is as follows:
6 samples of normal fertility seminal plasma, 6 samples of seminal plasma obtained by microscopical collection of patients without spermatozoon, 500 microliters of seminal plasma obtained by microscopical collection of patients without spermatozoon, and extraction of all small RNAs of seminal plasma (using a kit of mirVana of Seimeri Fei Co., Ltd.)TMmiRNA Isolation Kit, cat # AM1561) (total small RNAs less than 200 nt), establishing a sequencing library, carrying out UMI absolute quantitative small RNA sequencing by a BGI-500 sequencer, carrying out comparative analysis on sequencing data and a human miRNA database, and identifying all miRNAs (total 80) differentially expressed by seminal plasma of three groups of people. Through expression quantity analysis, expression abundance analysis and experimental verification, 4 stably expressed differential miRNAs (mir-34b, mir-34c, mir-3065 and mir-4446) are finally determined as final molecular markers.
The nucleotide sequences of the 4 miRNA markers are as follows:
mir-34b CAATCACTAACTCCACTGCCAT(SEQ ID NO.1)；
mir-34c AATCACTAACCACACGGCCAGG(SEQ ID NO.2)；
mir-3065TCAGCA CCAGGATATTGTTGGAG(SEQ ID NO.3)；
mir-4446CAGGGCTGGCAGTGACATGGGT(SEQ ID NO.4)。
example 2
Prejudging microscopic semen collection operation by using miRNA molecular marker
1. Establishment of miRNA panel
Collecting 40 samples (6 samples with normal fertility, 12 samples with azoospermia micro-semen collection and 22 samples with azoospermia patients incapable of obtaining azoospermia), extracting small RNA, performing reverse transcription by using a specific stem-loop primer, performing real-time quantitative PCR amplification on the four miRNAs, performing sequential logistic regression analysis on an experimental result by using R, establishing a machine learning model, and finally obtaining the miRNAPlanel.
For each sample, 10 microliters of system was prepared by taking 5 microliters of miRNA, adding 1 microliter of mixed stem-loop primer (concentration 50nM), 2 microliters of 5X reverse transcriptase buffer, 1 microliter of reverse transcriptase, and 1 microliter of nuclease-free water. The PCR instrument was set up as follows: 30 minutes at 16 ℃; 60 cycles of 20 ℃ 30s, 42 ℃ 30s, 50 ℃ 1 s; 85 ℃ for 5 min. Taking 5 microliter of the reverse transcription product to carry out pre-amplification PCR (pre-PCR) reaction, and the specific process is as follows: 1 microliter of forward primer mix (50 pm concentration), 0.5 microliter of reverse primer (100 pm concentration), 12.5 microliter of Q5 Hi-Fi PCR mix (NEB Corp.), nuclease-free water to make up to 25 microliter. The setting conditions of the PCR instrument are as follows: 30s at 98 ℃; 12 cycles of 98 ℃ for 10s, 68 ℃ for 30s, and 72 ℃ for 30 s; 72 ℃ for 5 min.
And (3) carrying out qPCR amplification after diluting the pre-amplification product by 25 times, wherein the specific experimental process comprises the following steps: 1 microliter of the diluted pre-amplification product, 1 microliter of 10pm forward primer, 1 microliter of 10pm reverse primer, 1 microliter of 10pm probe, 10 microliter of 2X probe PCR reagent (TAKARA) are taken, and the nuclease-free water is filled to 20 microliter. The qPCR instrument setup program was: 30s at 95 ℃; 40 cycles of 10s at 95 ℃; 60 ℃ for 1 min.
The specific stem-loop primer sequences are as follows:
the PCR amplification primer sequences are as follows:
the specific probe amplification primer sequences are as follows:
inputting the obtained CT value data into R, performing ordered logistic regression analysis, dividing 40 data into two groups of training and test, performing machine learning, and obtaining an equation (miRNA panel)
The resulting miRNA panel is shown below:
2. double-blind tests are carried out in 40 cases of clinical samples, patient seminal plasma miRNA is extracted, the specific stem-loop primer is used for reverse transcription according to the experimental process, then the pre-amplification primer is used for carrying out a pre-PCR (pre-PCR) experiment, then the specific probe is used for carrying out multiplex digital PCR (multiplex ddPCR), corresponding fluorescence values are obtained and substituted into the model equation, and probability values are obtained, wherein the probability values are the probability values for prejudging the success rate of the microscopic semen collection operation.
In this embodiment, a double-blind test is performed on 40 clinical samples, that is, the experimenter does not know the type of the clinical sample (semen presence/absence), and the 40 samples are detected according to the standard operation of the kit to obtain the results: 20 patients with azoospermia, 14 patients with azoospermia and 6 patients with normal fertility. The results of the microscopic semen collection operation of the 40 sample clinicians are as follows: there were 22 patients without sperm, 12 patients with sperm, and 6 patients with normal fertility.
The results of the experiment are shown in FIGS. 1 to 3.
FIG. 1 shows the qPCR results of 40 samples, F1-F6 are normal fertility samples, E1-E12 are microscopically sperm-obtained samples of patients without sperm, and N1-N22 are microscopically sperm-not-obtained samples of patients without sperm. The different colors represent that the expression quantity of the four miRNAs is from high to low, and each sample can be distinguished according to the expression quantity.
Figure 2 is the correlation between four mirnas. Var1-4 respectively represents mir-34b, mir-34c, mir-3065 and mir-4446, wherein the correlation coefficient of the mir-34b and the mir-34c is 0.61, and the mir-34b and the mir-34c are both prone to be highly expressed in a spermatogenic specimen, namely, if the two small RNAs are highly expressed, spermatogenesis is proved in a testis of a patient, and the rate of obtaining spermatozoa through microscopic spermatozoa is high; the correlation coefficient of mir-3065 and mir-4446 is 0.75, and both tend to be highly expressed in a spermatogenesis-free specimen, i.e., if the two small RNAs are highly expressed, it is proved that the patient has no spermatogenesis in the testis, and the rate of sperm collection by microscopic sperm is very low.
FIG. 3 is a ROC curve to assess the accuracy of each small RNA in predicting spermatogenic function. The closer the curve is to the upper left corner, the higher the prediction accuracy. The horizontal axis of the coordinate axis represents the false positive rate, and the vertical axis represents the true positive rate.
The obtained experimental result is compared with the microscopical semen collection operation result of a clinician, the identification rate of the patients without semen is 100 percent, and the misjudgment rate of the patients without semen is 14 percent. Namely, the coincidence rate of the azoospermia patient detected by the kit and the clinical operation result is 100%, but 2 cases of misjudgment exist, and the technical problem of operation of a clinician is not solved. The result proves that through preoperative kit detection, unnecessary traumatic detection and operation of an aspermatic patient can be avoided, resources are saved, efficiency is improved, important preoperative reference can be given to a surgeon, operation success rate is improved, no traumatic exists, and operation is simple and convenient.
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Claims (6)
1. A group of miRNA molecular markers for judging the success rate of microscopical semen collection surgery is characterized in that: the markers comprise miR-34b-3p, miR-34c-3p, miR-3065-3p and miR-4446-3 p.
2. Use of the miRNA molecular marker of claim 1 in the preparation of a kit for determining the success rate of microscopical semen collection surgery.
3. A primer pair for detecting the miRNA molecular marker set forth in claim 1, wherein: the method comprises the following specific steps:
for miR-34b-3p, the sequence of an upstream primer is shown as SEQ ID NO.9, the sequence of a downstream primer is shown as SEQ ID NO. 13;
for miR-34c-3p, the sequence of an upstream primer is shown as SEQ ID NO.10, the sequence of a downstream primer is shown as SEQ ID NO. 13;
for miR-3065-3p, the sequence of an upstream primer is shown as SEQ ID NO.11, the sequence of a downstream primer is shown as SEQ ID NO. 13;
for miR-4446-3p, the sequence of an upstream primer is shown as SEQ ID NO.12, the sequence of a downstream primer is shown as SEQ ID NO.13, and the sequence is shown as a universal primer.
4. Use of the primer pair of claim 3 in the preparation of a kit for determining microscopical semen collection surgery success rate.
5. A kit for judging the success rate of microscopical semen collection surgery is characterized in that: comprising the primer pair of claim 3.
6. The kit of claim 5, wherein: also comprises reverse transcription stem-loop primers, probes, reagents commonly used in PCR technology and reagents commonly used in immunohistochemical technology.
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| CN109136358A (en) * | 2017-06-27 | 2019-01-04 | 华中科技大学同济医学院生殖医学中心 | The reagent of remaining sperm and miRNA are in application wherein in antidiastole NOA patient's testis |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109136358A (en) * | 2017-06-27 | 2019-01-04 | 华中科技大学同济医学院生殖医学中心 | The reagent of remaining sperm and miRNA are in application wherein in antidiastole NOA patient's testis |
Non-Patent Citations (4)
| Title |
|---|
| 卞玉莹等: "精浆非编码小RNA与男性不育最新研究进展", 《中华男科学杂志》 * |
| 娄江涛等: "miR-34b在男性不育患者精浆中的表达及其临床意义", 《中国男科学杂志》 * |
| 王仪春等: "预测非梗阻性无精子症显微取精结局的相关标志物的研究现状", 《中华男科学杂志》 * |
| 赵文忠等: "非阻塞性无精症患者血清miRNA表达谱分析研究", 《重庆医学》 * |
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